Early Mouse Embryonic Development Research Articles

Lactate modulates zygotic genome activation through H3K18 lactylation rather than H3K27 acetylation

In spite of its essential role in culture media, the precise influence of lactate on early mouse embryonic development remains elusive. Previous studies have implicated lactate accumulation in medium affecting histone acetylation. Recent research has underscored lactate-derived histone lactylation as a novel epigenetic modification in diverse cellular processes and diseases. Our investigation demonstrated that the absence of sodium lactate in the medium resulted in a pronounced 2-cell arrest at the late G2 phase in embryos. RNA-seq analysis revealed that the absence of sodium lactate significantly impaired the maternal-to-zygotic transition (MZT), particularly in zygotic gene activation (ZGA). Investigations were conducted employing Cut&Tag assays targeting the well-studied histone acetylation and lactylation sites, H3K18la and H3K27ac, respectively. The findings revealed a noticeable reduction in H3K18la modification under lactate deficiency, and this alteration showed a significant correlation with changes in gene expression. In contrast, H3K27ac exhibited minimal correlation. These results suggest that lactate may preferentially influence early embryonic development through H3K18la rather than H3K27ac modifications.

P-115 GATAD2B is required for pre-implantation embryo development by delaying zygotic genome activation

Abstract Study question Major zygotic genome activation (ZGA) involving the activation of thousands of genes occurs at the late-2cell stage, but the regulatory mechanisms governing ZGA remain unclear. Summary answer Our results demonstrate that GATAD2B is essential for early embryonic development, in part through facilitating zygotic genome activation (ZGA). What is known already Acetylation modifications of histone tails promote transcriptional activation, and the maternal deletion of H4K16ac leads to failure in ZGA. GATAD2B is one of the core subunits of the Nucleosome remodeling and histone deacetylation complex(NuRD). Study design, size, duration Using mouse embryos as the research model, the study was conducted over a period of approximately two years to ensure a relatively small-scale investigation. Participants/materials, setting, methods We examined the dynamic expression of NURD complex components, including GATAD2B, during pre-implantation embryonic stages. GATAD2B expression was specifically reduced via microinjection of GATAD2B-siRNA during the zygote stage, revealing its impact on early embryonic development in mice. Zygotic genome activation (ZGA), morula compaction, and blastocyst lineage differentiation were investigated using immunofluorescence, western blot, and other techniques. Additionally, we employed RNA-seq, protein immunoprecipitation, and mass spectrometry to uncover underlying molecular mechanisms. Main results and the role of chance Our research has shown that GATAD2B exhibits specific nucleus localization and high protein expression from the late-2-cell to 8-cell. This intriguing phenomenon prompted us to investigate the relationship between GATAD2B and the ZGA. We discovered a distinctive pattern of GATAD2B, starting from the late 2-cell stage with nuclear localization. GATAD2B depletion resulted in defective embryonic development, including increased DNA damage at morula, decreased blastocyst formation rate and abnormal differentiation of ICM/TE lineages. Consistent with the delay during the cleavage stage, the transcriptome analysis of the 2-cell revealed inhibition of the cell cycle G2/M phase transition pathway. Furthermore, the GATAD2B proteomics data provided clear evidence of a certain association between GATAD2B and molecules involved in the cell cycle pathway. As hypothesized, GATAD2B-deficient 2-cell embryos exhibited abnormalities in ZGA during the maternal-to-embryonic transition, with lower expression of the major ZGA mark MERVL. Limitations, reasons for caution Given the limitations of antibody effectiveness and specificity required for the experiments, the exploration of molecular mechanisms is still ongoing. Wider implications of the findings Our findings have elucidated the impact of GATAD2B on early embryonic development through the regulation of zygotic genome activation (ZGA), providing a novel genetic explanation for clinical defects in early embryonic development. Trial registration number not applicable

O-077 Depletion of MFF from oocytes impairs mitochondrial dynamics, leading to inhibited oocyte maturation and early embryonic development in mice

Abstract Study question To investigate the effect of mitochondrial fission factor (MFF) on oocyte competence and female fertility using an oocyte-specific Mff knockout mouse model. Summary answer Oocyte-specific Mff deficiency hindered oocyte maturation and early embryonic development via regulation of mitochondrial dynamics, resulting in female subfertility. What is known already Emerging studies have demonstrated that mitochondrial dynamics play a vital role in oocyte maturation and early embryo development. MFF is one of the crucial proteins regulating mitochondrial dynamics. Our previous studies have indicated that Mff is necessary for oocyte competence and fertility using a global Mff knockout mouse model. However, further investigation is needed to determine whether the deletion of Mff in oocytes could affect oocyte competence and early embryonic development by regulating mitochondrial dynamics. Study design, size, duration The oocyte-specific Mff knockout (Mfffl/fl; Gdf9-Cre) mice were generated using the Cre-LoxP conditional knockout system. The 2-month-old female mice from each group (Mfffl/fl; Gdf9-Cre or Mfffl/fl, n = 6) were mated to assess the fecundity over 10 months. The maturation and mitochondrial dynamics of germinal vesicle (GV) oocytes were determined. The spindle and chromosome morphologies of ovulated oocytes were also analyzed. Additionally, the 2-cell embryos were collected and cultured in vitro to assess early embryonic development. Participants/materials, setting, methods Female mice from each group were mated with adult males to assess the fecundity. GV oocytes, ovulated oocytes, and 2-cell embryos were collected and cultured in vitro after superovulation, as indicated. Mitochondria, spindle, and chromosome were stained and then imaged using confocal microscopy. The microscopic morphology of mitochondria was assessed using electron microscopy. Main results and the role of chance The Mfffl/fl; Gdf9-Cre mice consistently exhibited reduced fertility with a significant decrease in litter size (3.00 vs. 5.42, p < 0.001), number of litters per female (4.33 vs. 7.50, p < 0.001), and number of pups per female (13.00 vs. 40.67, p < 0.001) compared to the Mfffl/fl mice. The numbers of GV oocytes (29.37 vs. 39.10, p < 0.05), ovulated oocytes (9.81 vs. 20.91, p < 0.001) and 2-cell embryos (3.50 vs. 10.17, p < 0.05) were significantly lower in the Mfffl/fl; Gdf9-Cre mice. The blastocyst (32.32% vs. 68.44%, p < 0.05) embryo development rate per 2-cell embryos was also significantly lower. In vitro maturation revealed that of Mfffl/fl; Gdf9-Cre GV oocytes had significantly lower rates of GV break-down (68.89% vs. 91.57%, p < 0.05) and polar body extrusion (42.76% vs. 81.87%, p < 0.05). The ratios of aberrant spindle (71.56% vs. 23.74%, p < 0.001) and misaligned chromosomes (71.82% vs. 23.14%, p < 0.01) in ovulated oocytes from Mfffl/fl; Gdf9-Cre mice were significantly increased. In terms of mitochondrial dynamics, the mitochondrial size (0.59 µm2 vs. 0.15µm2, p < 0.001) and the ratio (62.50% vs. 18.67%, p < 0.01) of abnormal mitochondrial distribution (unevenly aggregated) in Mfffl/fl; Gdf9-Cre oocytes significantly elevated. This study found that the Mff played a crucial role in oocyte maturation and early embryonic development. Limitations, reasons for caution Further research is needed to determine the downstream targets or pathways of Mff in oocytes. Wider implications of the findings These results shed light on the important role played by MFF in ensuring proper mitochondrial dynamics in oocytes and underscore the significance of this protein in female fertility. This study provides insights into potential therapeutic targets for infertility and related diseases in humans. Trial registration number not applicable

Disruption of early embryonic development in mice by polymethylmethacrylate nanoplastics in an oxidative stress mechanism

Polymethylmethacrylate (PMMA) has been used in many products, such as acrylic glass, and is estimated to reach 5.7 million tons of production per year by 2028. Thus, nano-sized PMMA particles in the environment are highly likely due to the weathering process. However, information on the hazards of nanoplastics, including PMMA in mammals, especially reproductive toxicity and action mechanism, is scarce. Herein, we investigated the effect of PMMA nanoplastics on the female reproductive system of mice embryos during pre-implantation. The treated plastic particles in embryos (10, 100, and 1000 μg/mL) were endocytosed into the cytoplasm within 30 min, and the blastocyst development and indices of embryo quality were significantly decreased from at 100 μg/mL. Likewise, the transfer of nanoplastic-treated embryos at 100 μg/mL decreased the morula implantation rate on the oviduct of pseudopregnant mice by 70%, calculated by the pregnant individual, and 31.8% by the number of implanted embryos. The PMMA nanoplastics at 100 μg/mL significantly increased the cellular levels of reactive oxygen species in embryos, which was not related to the intrinsic oxidative potential of nanoplastics. This study highlights that the nanoplastics that enter systemic circulation can affect the early stage of embryos. Thus, suitable action mechanisms can be designed to address nanoplastic occurrence.

Loss of KANSL3 leads to defective inner cell mass and early embryonic lethality.

e-Lysine acetylation is a prominent histone mark found at transcriptionally active loci. Among many lysine acetyl transferases, nonspecific lethal complex (NSL) members are known to mediate the modification of histone H4. In addition to histone modifications, the KAT8 regulatory complex subunit 3 gene (Kansl3), a core member of NSL complex, has been shown to be involved in several other cellular processes such as mitosis and mitochondrial activity. Although functional studies have been performed on NSL complex members, none of the four core proteins, including Kansl3, have been studied during early mouse development. Here we show that homozygous knockout Kansl3 embryos are lethal at peri-implantation stages, failing to hatch out of the zona pellucida. When the zona pellucida is removed in vitro, Kansl3 null embryos form an abnormal outgrowth with significantly disrupted inner cell mass (ICM) morphology. We document lineage-specific defects at the blastocyst stage with significantly reduced ICM cell number but no difference in trophectoderm cell numbers. Both epiblast and primitive endoderm lineages are altered with reduced cell numbers in null mutants. These results show that Kansl3 is indispensable during early mouse embryonic development and with defects in both ICM and trophectoderm lineages.

Abnormalities in pharyngeal arch-derived structures in SATB2-associated syndrome.

SATB2-associated syndrome (SAS, glass syndrome, OMIM#612313) is a neurodevelopmental autosomal dominant disorder with frequent craniofacial abnormalities including palatal and dental anomalies. To assess the role of Satb2 in craniofacial development, we analyzed mutant mice at different stages of development. Here, we show that Satb2 is broadly expressed in early embryonic mouse development including the mesenchyme of the second and third arches. Satb2-/- mutant mice exhibit microglossia, a shortened lower jaw, smaller trigeminal ganglia, and larger thyroids. We correlate these findings with the detailed clinical phenotype of four individuals with SAS and remarkable craniofacial phenotypes with one requiring mandibular distraction in childhood. We conclude that the mouse and patient data presented support less well-described phenotypic aspects of SAS including mandibular morphology and thyroid anatomical/functional issues.

Overexpression of KAT8 induces a failure in early embryonic development in mice

Embryo quality is strongly associated with subsequent embryonic developmental efficiency. However, the detailed function of lysine acetyltransferase 8 (KAT8) during early embryonic development in mice remains elusive. In this study, we reported that KAT8 played a pivotal role in the first cleavage of mouse embryos. Immunostaining results revealed that KAT8 predominantly accumulated in the nucleus throughout the entire embryonic developmental process. Kat8 overexpression (Kat8-OE) was correlated with early developmental potential of embryos to the blastocyst stage. We also found that Kat8-OE embryos showed spindle-assembly defects and chromosomal misalignment, and that Kat8-OE in embryos led to increased levels of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX by affecting the expression of critical genes related to mitochondrial respiratory chain and antioxidation pathways. Subsequently, cellular apoptosis was activated as confirmed by TUNEL (Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling) assay. Furthermore, we revealed that KAT8 was related to regulating the acetylation status of H4K16 in mouse embryos, and Kat8-OE induced the hyperacetylation of H4K16, which might be a key factor for the defective spindle/chromosome apparatus. Collectively, our data suggest that KAT8 constitutes an important regulator of spindle assembly and redox homeostasis during early embryonic development in mice.

Neddylation inhibition affects early embryonic development by disrupting maternal-to-zygotic transition and mitochondrial function in mice

Post-translational modifications (PTMs) are critical for early development in mice because early cleavage-stage embryos are characterized by transcriptional inactivity. Neddylation is an important ubiquitin-like PTM that regulates multiple biophysical processes. However, the exact roles of neddylation in regulating early embryonic development remain largely unknown. In the present study, we found that inhibition of neddylation by specific inhibitor MLN4924 led to severe arrest of early embryonic development. Transcriptomic analysis showed that neddylation inhibition changed the expression of 3959 genes at the 2-cell stage. Importantly, neddylation inhibition blocked zygotic genome activation and maternal mRNA degradation, thus disrupting the maternal-to-zygotic transition. Moreover, inhibition of neddylation induced mitochondrial dysfunction including aberrant mitochondrial distribution, decreased mitochondrial membrane potential, and reduced ATP content. Further analysis showed that inhibition of neddylation resulted in the accumulation of reactive oxygen species and superoxide anion, thereby resulting in oxidative stress and severe DNA damage at the 2-cell stage. Overall, this study demonstrates that neddylation is vital for early embryonic development in mice. Our findings suggest that proper neddylation regulation is essential for the timely inter-stage transition during early embryonic development.

Lineage motifs as developmental modules for control of cell type proportions

In multicellular organisms, cell types must be produced and maintained in appropriate proportions. One way this is achieved is through committed progenitor cells or extrinsic interactions that produce specific patterns of descendant cell types on lineage trees. However, cell fate commitment is probabilistic in most contexts, making it difficult to infer these dynamics and understand how they establish overall cell type proportions. Here, we introduce Lineage Motif Analysis (LMA), a method that recursively identifies statistically overrepresented patterns of cell fates on lineage trees as potential signatures of committed progenitor states or extrinsic interactions. Applying LMA to published datasets reveals spatial and temporal organization of cell fate commitment in zebrafish and rat retina and early mouse embryonic development. Comparative analysis of vertebrate species suggests that lineage motifs facilitate adaptive evolutionary variation of retinal cell type proportions. LMA thus provides insight into complex developmental processes by decomposing them into simpler underlying modules.

Pivotal role for long noncoding RNAs in zygotic genome activation in mice.

Vertebrate life begins with fertilization, and then the zygote genome is activated after transient silencing, a process termed zygotic genome activation (ZGA). Despite its fundamental role in totipotency and the initiation of life, the precise mechanism underlying ZGA initiation remains unclear. The existence of minor ZGA implies the possible critical role of noncoding RNAs in the initiation of ZGA. Here, we delineate the expression profile of long noncoding RNAs (lncRNAs) in early mouse embryonic development and elucidate their critical role in minor ZGA. Compared with protein-coding genes (PCGs), lncRNAs exhibit a stronger correlation with minor ZGA. Distinct H3K9me3 profiles can be observed between lncRNA genes and PCGs, and the enrichment of H3K9me3 before ZGA might explain the suspended expression of major ZGA-related PCGs despite possessing PolII pre-configuration. Furthermore, we identified the presence of PolII-enriched MuERV-L around the transcriptional start site of minor ZGA-related lncRNAs, and these repeats are responsible for the activation of minor ZGA-related lncRNAs and subsequent embryo development. Our study suggests that MuERV-L mediates minor ZGA lncRNA activation as a critical driver between epigenetic reprogramming triggered by fertilization and the embryo developmental program, thus providing clues for understanding the regulatory mechanism of totipotency and establishing bona fide totipotent stem cells.

Instantaneous Manipulation: Dynamic Regulation of Eomes in Early Mouse Embryonic Development

Instantaneous Manipulation: Dynamic Regulation of Eomes in Early Mouse Embryonic Development

FGFR1 variants contributed to families with tooth agenesis

BackgroundTooth agenesis is a common dental anomaly that can substantially affect both the ability to chew and the esthetic appearance of patients. This study aims to identify possible genetic factors that underlie various forms of tooth agenesis and to investigate the possible molecular mechanisms through which human dental pulp stem cells may play a role in this condition.ResultsUsing whole-exome sequencing of a Han Chinese family with non-syndromic tooth agenesis, a rare mutation in FGFR1 (NM_001174063.2: c.103G > A, p.Gly35Arg) was identified as causative and confirmed by Sanger sequencing. Via GeneMatcher, another family with a known variant (NM_001174063.2: c.1859G > A, p.Arg620Gln) was identified and diagnosed with tooth agenesis and a rare genetic disorder with considerable intrafamilial variability. Fgfr1 is enriched in the ectoderm during early embryonic development of mice and showed sustained low expression during normal embryonic development of Xenopus laevis frogs. Functional studies of the highly conserved missense variant c.103G > A showed deleterious effects. FGFR1 (c.103G > A) was overexpressed compared to wildtype and promoted proliferation while inhibiting apoptosis in HEK293 and human dental pulp stem cells. Moreover, the c.103G > A variant was found to suppress the epithelial-mesenchymal transition. The variant could downregulate ID4 expression and deactivate the TGF-beta signaling pathway by promoting the expression of SMAD6 and SMAD7.ConclusionOur research broadens the mutation spectrum associated with tooth agenesis and enhances understanding of the underlying disease mechanisms of this condition.

FOXG1 is involved in mouse ovarian functions and embryogenesis

TGF-β superfamily has long been demonstrated to be essential for folliculogenesis and luteinization. Forkhead box G1 (FOXG1, also known as BF1), a member of the FOX family and an inhibitor of TGF-β signaling pathway, is a nucleocytoplasmic transcription factor that is essential for forebrain development. FOXG1 is involved in neurodevelopment and cancer pathology, however, little is known about the role of FOXG1 in reproduction. In this study, the spatiotemporal expression pattern of FOXG1 was examined during early mouse oocyte and embryonic development and its role during corpora luteum (CL) formation was further elucidated. The results showed that FOXG1 is localized in oocytes, theca cells (TCs) and CLs. After fertilization, FOXG1 is expressed at all stages during early embryogenesis, from zygotes to blastocysts. Following gonadotropin administration in immature mice, the expression of Foxg1 significantly increased along with steroidogenic genes, including Star, Hsd3β, Cyp11a1, as well as Cyp17a1 and Cyp19a1. The latter two first increased after pregnant mare serum gonadotropin stimulation, then decreased in response to hCG treatment. In addition, silencing of Foxg1 significantly reduced the concentration of testosterone and estrogen in cultured primary granulosa cells (GCs) and TCs (P < 0.05). Mechanistic studies demonstrated that the expression level of genes that are critical in estrogen synthesis were significantly reduced after Foxg1 silencing, including Cyp17a1 and Cyp19a1. In conclusion, FOXG1 is expressed in a stage-specific manner during folliculogenesis and embryogenesis and exerts a regulatory influence on testosterone and estrogen synthesis.

Maternal TDP-43 interacts with RNA Pol II and regulates zygotic genome activation

Zygotic genome activation (ZGA) is essential for early embryonic development. However, the regulation of ZGA remains elusive in mammals. Here we report that a maternal factor TDP-43, a nuclear transactive response DNA-binding protein, regulates ZGA through RNA Pol II and is essential for mouse early embryogenesis. Maternal TDP-43 translocates from the cytoplasm into the nucleus at the early two-cell stage when minor to major ZGA transition occurs. Genetic deletion of maternal TDP-43 results in mouse early embryos arrested at the two-cell stage. TDP-43 co-occupies with RNA Pol II as large foci in the nucleus and also at the promoters of ZGA genes at the late two-cell stage. Biochemical evidence indicates that TDP-43 binds Polr2a and Cyclin T1. Depletion of maternal TDP-43 caused the loss of Pol II foci and reduced Pol II binding on chromatin at major ZGA genes, accompanied by defective ZGA. Collectively, our results suggest that maternal TDP-43 is critical for mouse early embryonic development, in part through facilitating the correct RNA Pol II configuration and zygotic genome activation.

Acute exposure of triclocarban affects early embryo development in mouse through disrupting maternal-to-zygotic transition and epigenetic modifications

Triclocarban (TCC) is a broad-spectrum antibacterial agent used globally, and high concentrations of this harmful chemical exist in the environment. The human body is directly exposed to TCC through skin contact. Moreover, TCC is also absorbed through diet and inhaled through breathing, which results in its accumulation in the body. The safety profile of TCC and its potential impact on human health are still not completely clear; therefore, it becomes imperative to evaluate the reproductive toxicity of TCC. Here, we explored the effect of TCC on the early embryonic development of mice and its associated mechanisms. We found that acute exposure of TCC affected the early embryonic development of mice in a dose-dependent manner. Approximately 7600 differentially expressed genes (DEGs) were obtained by sequencing the transcriptome of 2-cell mouse embryos; of these, 3157 genes were upregulated and 4443 genes were downregulated in the TCC-treated embryos. GO and KEGG analysis revealed that the enriched genes were mainly involved in redox processes, RNA synthesis, DNA damage, apoptosis, mitochondria, endoplasmic reticulum, Golgi apparatus, cytoskeleton, peroxisome, RNA polymerase, and other components or processes. Moreover, the Venn analysis showed that the zygotic genome activation (ZGA) was affected and the degradation of maternal effector genes was inhibited. TCC induced changes in the epigenetic modification of 2-cell embryos. The level of DNA methylation increased significantly. Further, the levels of H3K27ac, H3K9ac, and H3K27me3 histone modifications decreased significantly, whereas those of H3K4me3 and H3K9me3 modifications increased significantly. Additionally, TCC induced oxidative stress and DNA damage in the 2-cell embryos. In conclusion, acute exposure of TCC affected early embryo development, destroyed early embryo gene expression, interfered with ZGA and maternal gene degradation, induced changes in epigenetic modification of early embryos, and led to oxidative stress and DNA damage in mouse early embryos.

Knockdown of Csnk1a1 results in preimplantation developmental arrest in mice

Casein kinase 1, alpha 1 (CSNK1A1), is a member of the highly conserved serine/threonine protein kinase family. This study was established to analyze the expression and localization of CSNK1A1 and its function in early embryonic development in mice. Csnk1a1 mRNA and protein are expressed in multiple mouse tissues including the ovary. After ovulation and fertilization, Csnk1a1 mRNA and protein were detected in preimplantation embryos and their expression was highest in two-cell-stage embryos. CSNK1A1 protein was also mainly localized in the cytoplasm of preimplantation embryos. Moreover, knockdown of Csnk1a1 in zygotes led to a significant decrease in the rate of blastocyst formation. Furthermore, treatment of zygotes with the CSNK1A1-specific inhibitor D4476 also resulted in embryonic developmental arrest. These results provide the first evidence for a novel function of CSNK1A1 in early embryonic development in mice.

Translation regulatory factor BZW1 regulates preimplantation embryo development and compaction by restricting global non-AUG Initiation

Protein synthesis is an essential step in gene expression during the development of mammalian preimplantation embryos. This is a complex and highly regulated process. The accuracy of the translation initiation codon is important in various gene expression programs. However, the mechanisms that regulate AUG and non-AUG codon initiation in early embryos remain poorly understood. BZW1 is a key factor in determining the mRNA translation start codon. Here, we show that BZW1 is essential for early embryonic development in mice. Bzw1-knockdown embryos fail to undergo compaction, and show decreased blastocyst formation rates. We also observe defects in the differentiation capacity and implantation potential after Bzw1 interference. Further investigation revealed that Bzw1 knockdown causes the levels of translation initiation with CUG as the start codon to increase. The decline in BZW1 levels result in a decrease in protein synthesis in preimplantation embryos, whereas the total mRNA levels are not altered. Therefore, we concluded that BZW1 contributes to protein synthesis during early embryonic development by restricting non-AUG translational initiation.

Multi-Level Control of the ATM/ATR-CHK1 Axis by the Transcription Factor E4F1 in Triple-Negative Breast Cancer.

E4F1 is essential for early embryonic mouse development and for controlling the balance between proliferation and survival of actively dividing cells. We previously reported that E4F1 is essential for the survival of murine p53-deficient cancer cells by controlling the expression of genes involved in mitochondria functions and metabolism, and in cell-cycle checkpoints, including CHEK1, a major component of the DNA damage and replication stress responses. Here, combining ChIP-Seq and RNA-Seq approaches, we identified the transcriptional program directly controlled by E4F1 in Human Triple-Negative Breast Cancer cells (TNBC). E4F1 binds and regulates a limited list of direct target genes (57 genes) in these cells, including the human CHEK1 gene and, surprisingly, also two other genes encoding post-transcriptional regulators of the ATM/ATR-CHK1 axis, namely, the TTT complex component TTI2 and the phosphatase PPP5C, that are essential for the folding and stability, and the signaling of ATM/ATR kinases, respectively. Importantly, E4F1 also binds the promoter of these genes in vivo in Primary Derived Xenograft (PDX) of human TNBC. Consequently, the protein levels and signaling of CHK1 but also of ATM/ATR kinases are strongly downregulated in E4F1-depleted TNBC cells resulting in a deficiency of the DNA damage and replicative stress response in these cells. The E4F1-depleted cells fail to arrest into S-phase upon treatment with the replication-stalling agent Gemcitabine, and are highly sensitized to this drug, as well as to other DNA-damaging agents, such as Cisplatin. Altogether, our data indicate that in breast cancer cells the ATM/ATR-CHK1 signaling pathway and DNA damage-stress response are tightly controlled at the transcriptional and post-transcriptional level by E4F1.

WDR36 Safeguards Self-Renewal and Pluripotency of Human Extended Pluripotent Stem Cells.

Extended pluripotent stem cells (EPS cells) have unlimited self-renewal ability and the potential to differentiate into mesodermal, ectodermal, and endodermal cells. Notably, in addition to developing the embryonic (Em) lineages, it can also make an effective contribution to extraembryonic (ExEm) lineages both in vitro and in vivo. However, multiple mysteries still remain about the underlying molecular mechanism of EPS cells’ maintenance and developmental potential. WDR36 (WD Repeat Domain 36), a protein of 105 kDa with 14 WD40 repeats, which may fold into two β-propellers, participates in 18sRNA synthesis and P53 stress response. Though WDR36 safeguards mouse early embryonic development, that is, homozygous knockout of WDR36 can result in embryonic lethality, what role does WDR36 plays in self-renewal and differentiation developmental potential of human EPS cells is still a subject of concern. Here, our findings suggested that the expression of WDR36 was downregulated during human hEPS cells lost self-renewal. Through constructing inducible knockdown or overexpressing WDR36-human EPS cell lines, we found that WDR36 knockdown disrupted self-renewal but promoted the mesodermal differentiation of human EPS cells; however, overexpressing of WDR36 had little effect. Additionally, P53 inhibition could reverse the effects of WDR36 knockdown, on both self-renewal maintenance and differentiation potential of human EPS cells. These data implied that WDR36 safeguards self-renewal and pluripotency of human EPS cells, which would extend our understanding of the molecular mechanisms of human EPS cells’ self-renewal and differentiation.

Mitochondrial Calcium Disorder Affects Early Embryonic Development in Mice through Regulating the ERK/MAPK Pathway.

The homeostasis of mitochondrial calcium ([Ca2+]mt) in oocytes plays a critical role in maintaining normal reproductive cellular progress such as meiosis. However, little is known about the association between [Ca2+]mt homeostasis and early embryonic development. Two in vitro mouse MII oocyte models were established by using a specific agonist or inhibitor targeting mitochondrial calcium uniporters (MCU) to upregulate or downregulate [Ca2+]mt concentrations. The imbalance of [Ca2+]mt in MII oocytes causes mitochondrial dysfunction and morphological abnormity, leading to an abnormal spindle/chromosome structure. Oocytes in drug-treated groups are less likely to develop into blastocyst during in vitro culture. Abnormal [Ca2+]mt concentrations in oocytes hindered epigenetic modification and regulated mitogen-activated protein kinase (MAPK) signaling that is associated with gene expression. We also found that MAPK/ERK signaling is regulating DNA methylation in MII oocytes to modulate epigenetic modification. These data provide a new insight into the protective role of [Ca2+]mt homeostasis in early embryonic development and also demonstrate a new mechanism of MAPK signaling regulated by [Ca2+]mt that influences epigenetic modification.